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Page 14 of 20Rai et al. • C. roseus Geranyl Diphosphate Synthasesinto IDS-b clade that includes three angiosperm GPPSs fromArabidopsis (AtGPPS), L. esculentum (LeGPS), and Q. rubur(QrIDS1), and one gymnosperm GPPS from P. abies (PaIDS3),which produced FPP and or GGPP in addition to GPP (Bouvieret al., 2000; van Schie et al., 2007; Schmidt and Gershenzon,2008). AtGPPS was recently shown to be a PPPS in contrast tothe earlier report (Hsieh et al., 2011). However, CrGPPS fromthe same IDS-b clade produced only GPP and showed mitochondriallocalization (Figure 10 and Figure 11). Interestingly,prediction analyses revealed that, like CrGPPS, the GPPSs ofA. grandis (AgGPPS1) of clade-IDSa2 and all proteins of cladeIDS-b seem to be localized to mitochondria (SupplementalTable 1). This suggested the possible role of IDS-a2 and IDS-bclade of enzymes in associated terpene biosynthesis pathwaysin plant cell. The IDS-c1 is represented by inactive SSU-I class ofheteromoeric GPPS, whereas an unusual homomeric GPPS fromorchid (P. bellina) lacking a DD(X) 2–4 D motif with close relatednessto SSUs has been clustered as IDS-c2 clade (Hsiao et al.,2008). The IDS-c3 clade contains a unique A. thaliana protein(AtSSU-II) with two CxxxC motifs and a FARM motif but lackinga SARM motif (Wang and Dixon, 2009) (Figure 11). AtSSU-IIshowed no activity but modified the chain length specificity ofAtGGPPS11 and HlGPPS.LSU, which is similar to the functionof other characterized SSUs (Wang and Dixon, 2009). Our phylogeneticanalysis demonstrated that IDS-c clade, consistingof members of IDS-c1, IDS-c2, and IDS-c3, could have evolvedfrom a common ancestor in which a homodimeric GPPS fallinginto IDS-c2 clade is active whereas SSU-I (IDS-c1) and SSU-II (IDSc3)are catalytically inactive. This could be due to the exceptionallyhigh speciation rate of orchids (Tsai and Chen, 2006)that would have altered the inactive SSU by modification ofa few amino acids, resulting in the required GPPS activity. Thisis evident with the presence of an alternate EAEVE motif ofPbGPPS, which is located in the equivalent position of SARM(Hsiao et al., 2008). The significance of presence of SSU-II inplants is currently unknown but a systematic characterizationof this class in different plants should provide the evidence forits role in plant metabolism.ConclusionsThe study of complex MIA biosynthesis and its regulationremains a great challenge in plant secondary metabolism. Thefunctional characterization of CrGPPSs in this study providedinsights into the formation of GPP in C. roseus. The CrGPPS.LSUmight have evolved to produce both GPP and GGPP in C. roseusto maintain relative amounts of precursors for both primary aswell as secondary metabolism. The plastid targeted CrGPPS.SSUand CrGPPS.LSU form active heteromeric GPPS to catalyze efficientproduction of GPP in vivo and provide better availability ofthis substrate to geraniol synthase, whereas homomeric CrGPPSmight be involved in mitochondrial isoprenoid biosynthesisfor the production of ubiquinones. The temporal and spatialexpression analysis of CrGPPS genes and transient overexpressionimplies that CrGPPS.SSU could function in coordinatedredirection of the metabolic flux, thereby acting as a primaryregulator to a certain extent in MIA biosynthesis. Takentogether, our results suggest that, under normal conditions, abasal level of the GPP pool is provided by both CrGPPS.LSU (abifunctional G(G)PPS) and heteromeric CrGPPS.LSU/CrGPPS.SSUtowards MIA formation in C. roseus. However, under biotic/abioticstress, the induced expression of SSU could result in an elevatedlevel of GPPS activity by LSU and SSU interaction, therebyincreasing the GPP pool towards MIA biosynthesis.METHODSPlant Material and MeJA TreatmentC. roseus cv. Dhawal (National Gene Bank, CSIR–CIMAP, India)plants were grown under normal greenhouse conditions.For MeJA treatments, 95% pure MeJA (Sigma-Aldrich) wasadded to a 5% (w/v) sucrose solution to a final concentrationof 200 mM. Excised leaves of the third developmental stagewere placed in a Petri plate containing the MeJA/sucrose orDMSO (the MeJA solvent) as a control. Samples were collectedat 0, 1, 4, 8, and 12 h, and stored at –80°C until further use.Chemicals and Radiochemicals[1- 3 H]-IPP (1.85 MBq) was purchased from AmericanRadiolabeled Chemicals (www.arcincusa.com). Unlabeled IPP,DMAPP, GPP, and FPP were purchased from Echelon ResearchLaboratories (www.echelon-inc.com). Terpenoid standardsand reagents were purchased from Sigma-Aldrich (www.sigmaaldrich.com)unless otherwise noted.RNA Isolation and cDNA SynthesisAbout 100 mg of tissue was ground in liquid nitrogen andtotal RNA was extracted using a Spectrum TM Plant Total RNAKit (Sigma-Aldrich, USA) according to the manufacturer’sinstructions. To amplify the 3′ end of CrGPPS.SSU, 3′ RACEcDNA was prepared with 5 µg total RNA using a SuperscriptIII RT Module (Invitrogen). Then, 3′ RACE PCR was carried outusing a GeneRacer TM Kit (Invitrogen) with 3′ RACE cDNA alongwith gene-specific reverse and nested primers and GeneRacer3′ and 3′ nested primers (Supplemental Table 2). The amplifiedfragment was cloned into pJET1.2/ vector and the sequencewas confirmed by nucleotide sequencing. cDNA for amplifyingopen reading frames of CrGPPSs and for real-time PCR analysiswas prepared by using a RevertAid H Minus First StrandcDNA Synthesis Kit (Fermentas International Inc., Canada).For real-time qRT–PCR, the DNA-free total RNA (5 μg) wasused for first-strand cDNA synthesis with Oligo(dT) 18 primersusing RevertAid H Minus Reverse Transcriptase. A linear rangeof cDNA was used for real-time PCR. Real-time qRT–PCR wasperformed in a 96-well plate using the Applied Biosystems7900HT Fast Real-Time PCR System (PE Applied Biosystems,www.appliedbiosystems.com) with SYBR green fluorescentdye using respective forward and reverse primers for eachgene (Supplemental Table 2). Fold change differences in geneDownloaded from http://mplant.oxfordjournals.org/ at Central Inst. Of Medicinal & Aromatic Plants (Cimap) on July 17, 2013
Rai et al. • C. roseus Geranyl Diphosphate Synthases Page 15 of 20expression were analyzed using the comparative cycle threshold(Ct) method (Applied Biosystems). Each data point representsthe mean of two independent biological replicates andthree technical replicates. Real-time qRT–PCR conditions wereas follows: 94°C for 10 min for one cycle, followed by 40 cyclesof 94°C for 15 s, 54°C for 15 s, and 72°C for 15 s.Phylogenetic AnalysisThe deduced amino acid sequence were aligned using MEGA4.0 (Tamura et al., 2007) with default settings. A phylogenetictree was constructed with the neighbor-joining method usingdefault settings of MEGA (Tamura et al., 2007). Bootstrap valueswere calculated from 1000 replicates. Shading of aminoacid sequence alignment was done by BOXSHADE 3.21 version(www.ch.embnet.org/software/BOX_form.html).Cloning of GPPS Genes and Generation ofOverexpression ConstructsThe open reading frame of each CrGPPS was amplified witha forward and a reverse primer consisting of the primers thatincluded a start and stop codon along with restriction sites forcloning into pET28a vector. The primer combinations used arementioned in Supplemental Table 2. PCR was performed usingPlatinum Taq DNA polymerase High Fidelity (Invitrogen) and PCRproducts were gel-purified using a gel extraction kit (Fermentas).PCR products were cloned into pJET 1.2/blunt cloning vector(CloneJET PCR cloning kit, Fermentas) and transformed intoE. coli XL1 for plasmid amplification. After restriction digestionand gel extraction, resulting fragments were sub-cloned into theexpression vector pET28a (Novagen) downstream and in-frameof (His) 6 -tag or pET32a for non-(His) 6 -tag clones. Positive cloneswere selected and the resulting constructs were confirmed byrestriction digestion and nucleotide sequencing.Expression and Purification of Recombinant ProteinsFor functional expression, E. coli Rosetta-2 competent cellswere transformed with recombinant plasmids and pET28a/pET32a lacking an insert (control). Induction, harvesting,and protein purification by affinity chromatography onnickel-nitrilotriacetic acid agarose (Bio-Rad, www.bio-rad.com) were performed as described by Nagegowda et al.(2008). Briefly, a single colony was used to inoculate 25 mlLuria-Bertani medium with 37 mg ml –1 chloramphenicol and50 mg ml –1 ampicillin (for pET32a constructs) or 50 mg ml –1kanamycin (for pET28a constructs) or with both antibiotics(for co-expression of both pET32a/pET28a constructs), whichwere grown overnight at 37°C. Five ml of these cultures weretransferred to 1000 ml of the same medium and continued togrow at 37°C until an absorbance of 0.5 at OD 600 nm . Cultureswere then induced by the addition of isopropyl-1-thio-β-Dgalactopyranoside(IPTG) to a final concentration of 0.4 mMand grown for an additional 18 h at 18°C. Protein purificationby affinity chromatography on nickel-nitrilotriacetic (Ni-NTA)acid agarose (Bio-Rad) was performed. Protein concentrationwas determined using the Bradford method (Bradford, 1976).Prenyltransferase Assays and Product IdentificationPrenyltransferase assays were performed as described previously(Orlova et al., 2009) except for the use of 40 μM [1- 3 H]-IPPinstead of 14 C-IPP. The reaction was performed in a final volumeof 100 μl containing 40 μM[1- 3 H]-IPP (50 mCi mmol –1 )and 40 μM DMAPP in assay buffer (25 mM MOPSO, pH 7.0,10% [v/v] glycerol, 2 mM DTT, and 10 mM MgCl 2 ). For theidentification of reaction products, larger-scale assays wereperformed in a final volume of 200 μl containing 80 μM-IPPand 80 μM DMAPP in assay buffer (25 mM MOPSO, pH 7.0,10% [v/v] glycerol, 2 mM DTT, and 10 mM MgCl 2 ). Assays wereperformed for 6 h at 30°C. To stop the assay and hydrolyze alldiphosphate esters (including un-reacted substrate as well asproducts), 200 µl of a solution containing 2 units of bovineintestine alkaline phosphatase (18 units mg –1 ; Sigma-Aldrich)and 2 units of potato apyrase (25.2 units mg –1 ; Sigma-Aldrich)in 0.2 M Tris-HCl, pH 9.5, were added to samples followedby overnight incubation at 30°C. After enzymatic hydrolysis,the resulting prenyl alcohols were extracted with 1 ml hexaneand the hexane fraction was concentrated to 25 µl. The productswere separated on reversed-phase TLC plates (TLC Silicagel 60 RP-18 F 254 S; MERCK, Germany). Chromatography wasperformed using a methanol:water (95:5 v/v) mobile phase,and spots were visualized by exposure of TLC plates to iodinevapor. Triplicate assays were performed for all data points.Enzyme assays were performed with one of following cationspresent in the assay buffer at final concentrations of 10 mM:Mg 2+ , Mn 2+ , or K + . All results represent an average of threeindependent assays.In Vivo Genetic Complementation AssayThe pACCAR25ΔcrtE (Kainou et al., 1999) which contain thegene cluster crtX, crtY, crtI, crtB, and crtZ encoding carotenoidbiosynthetic enzymes except crtE encoding GGPPSwere used for determining in vivo GGPPS activity. ThepET28a–CrGPPS constructs or HsGGPPS/pBH (as positive controls)and pACCAR25ΔcrtE were co-transformed into E. coliBL21 (DE3) with respective antibiotics (chloramphenicol forpACCAR25ΔcrtE; ampicillin for HsGGPPS and pBH; kanamycinfor pET28a construct of CrGPPS.LSU, CrGPPS.SSU, andCrGPPS) and plated on LB plates to grow overnight at 37°C.A single colony from plates containing transformed colonieswas picked and used for growth and induction as mentionedin the earlier section. To quantify carotenoids, pellets wereobtained after centrifugation and dissolved in 90% (v/v) acetoneto extract pigments. The concentration of carotenoidwas determined by absorption at 450 nm using a Bio-RadSmartSpec Spectrophotometer (Chang et al., 2010).Transient Overexpression of AmGPPS.SSU in C. roseusLeaves by AgroinfiltrationThe plasmid pEF1.LIS–AmGPPS.SSU (Orlova et al., 2009) containingthe coding region of snapdragon (A. majus) GPPS.SSU under C. breweri LIS promoter (1038 bp) (Orlova et al.,Downloaded from http://mplant.oxfordjournals.org/ at Central Inst. Of Medicinal & Aromatic Plants (Cimap) on July 17, 2013
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